Preventing or treating viral infection using an inhibitor of the lsd1 protein, a mao inhibitor or an inhibitor of lsd1 and a mao inhibitor

ABSTRACT

An embodiment of the invention provides preventing or treating a viral infection of a host, comprising administering to the host an effective amount of an inhibitor of the protein LSD1 and/or a monoamine oxidase inhibitor. Another embodiment of the invention provides preventing or treating reactivation of a virus after latency in a host, comprising administering to the host an effective amount of an inhibitor of the protein LSD1 and/or a monoamine oxidase inhibitor. Another embodiment of the invention provides preventing or treating a viral infection in a mammal that has undergone, is undergoing, or will undergo an organ or tissue transplant, comprising administering to the mammal an effective amount of an inhibitor of the protein LSD1 and/or a monoamine oxidase inhibitor before, during, and/or after the organ or tissue transplant. The viral infection may be due to a herpesvirus, such as herpes simplex virus type 1 (HSV-1) or type 2 (HSV-2), varicella zoster virus (VZV), or cytomegalovirus (CMV). The viral infection may also be due to an adenovirus, including types 1-5.

CROSS-REFERENCE TO RELATED APPLICATIONS

This patent application claims the benefit of U.S. Provisional PatentApplication Nos. 61/083,304, filed Jul. 24, 2008 and 61/111,019, filedNov. 4, 2008, which are each incorporated by reference.

SEQUENCE LISTING

Incorporated by reference in its entirety herein is a nucleotide/aminoacid sequence listing submitted concurrently herewith.

BACKGROUND OF THE INVENTION

Methylation of chromatin, a reversible modification mediated by histonemethyl-transferases and demethylases, is a significant component ofcellular transcriptional regulation. Such chromatin modifications alsoimpact invading viral pathogens that rely upon the host celltranscriptional apparatus. During infection of viruses, the assembly andmodification of chromatin on the viral genomes has the potential todetermine the progression of lytic infection as well as controlrecurrent latency-reactivation cycles.

A need continues to exist for methods of preventing or treating a viralinfection of a host.

BRIEF SUMMARY OF THE INVENTION

An embodiment of the invention provides a method of preventing ortreating a viral infection of a host, comprising administering to thehost an effective amount of an inhibitor of the protein LSD1 and/or amonoamine oxidase inhibitor.

Another embodiment of the invention provides a method of preventing ortreating reactivation of a virus after latency in a host, comprisingadministering to the host an effective amount of an inhibitor of theprotein LSD1 and/or a monoamine oxidase inhibitor.

Another embodiment of the invention provides a method of preventing ortreating a viral infection in a mammal that has undergone, isundergoing, or will undergo an organ or tissue transplant, comprisingadministering to the mammal an effective amount of an inhibitor of theprotein LSD1 and/or a monoamine oxidase inhibitor before, during, and/orafter the organ or tissue transplant.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows treatment of varicella zoster virus (VZV)-infected cellswith monoamine oxidase inhibitors resulted in a dose dependent decreasein viral mRNA and IE proteins. The data were generated using qRT-PCRanalysis of the IE62 and control (s15, actin) mRNA levels in cellsinfected with VZV for 4 hrs in the presence of increasing amounts ofPargyline (Left panel) or Tranylcypromine (TCP, Right panel). Theresults are represented as the percent of levels in dimethyl sulfoxide(DMSO) treated cells.

FIG. 2 shows treatment of herpes simplex virus (HSV)-infected cells withmonoamine oxidase inhibitors resulted in a dose dependent decrease inviral mRNA and IE proteins. These data were generated using qRT-PCRanalyses of mRNA levels of HSV IE genes and control s15 in cells treatedwith selected concentrations of Pargyline or Tranylcypromine (TCP). ThemRNA levels are graphically represented as the percent of mRNA levels incontrol DMSO treated cells.

FIG. 3 shows a decrease in viral yield in HSV latently infected miceupon administration of TCP. The data are viral yields from a time courseof explanted trigeminal ganglia of latently infected mice in thepresence or absence of 2 mM TCP for 2 days or 4 days; n=6 for eachsample set. In the absence of TCP, DMSO was used as the controltreatment vehicle.

FIG. 4 shows a decrease in viral yield in HSV latently infected miceupon administration of TCP using a direct paired analysis in which eachhalf of a latently infected ganglia was explanted in the presence andabsence of TCP. The data are viral yield from paired explanted gangliaof latently infected mice in the presence or absence of 2 mM TCP for 2days; n=16 for each sample set. In the absence of TCP, DMSO was used asthe control treatment vehicle.

FIG. 5 shows inhibition of HSV reactivation from latency at variouslevels of TCP using viral yield from explanted trigeminal ganglia oflatently infected mice in the presence or absence of variousconcentrations of TCP for 2 days. In the absence of TCP, DMSO was usedas the control treatment vehicle.

FIG. 6 shows that reversal of TCP inhibition results in HSVreactivation. Viral yields from explanted trigeminal ganglia of latentlyinfected mice are shown in the absence or presence of 2 mM TCP for 2days. In the absence of TCP, DMSO was used as the control treatmentvehicle. A portion of the TCP treated samples were then incubated in theabsence of drug (DMSO) for 3 days (TCP-R).

FIG. 7 shows dose-dependent repression of adenovirus E1A expression byTCP, whereas the expression of the control tubulin is largelyunaffected.

DETAILED DESCRIPTION OF THE INVENTION

An embodiment of the invention provides a method of preventing ortreating a viral infection of a host, comprising administering to thehost an effective amount of an inhibitor of the protein LSD1 and/or amonoamine oxidase inhibitor.

Another embodiment of the invention provides a method of preventing ortreating reactivation of a virus after latency in a host, comprisingadministering to the host an effective amount of an inhibitor of theprotein LSD1 and/or a monoamine oxidase inhibitor.

Another embodiment of the invention provides a method of preventing ortreating a viral infection of a host, comprising administering to thehost an effective amount of an inhibitor of the protein LSD1 and/or amonoamine oxidase inhibitor, wherein the administration of theinhibitor(s) prevents or treats the viral infection.

Another embodiment of the invention provides a method of preventing ortreating reactivation of a virus after latency in a host, comprisingadministering to the host an effective amount of an inhibitor of theprotein LSD1 and/or a monoamine oxidase inhibitor, wherein theadministration of the inhibitor(s) prevents or treats the viralreactivation.

A “host” may be considered a single cell, a tissue, an organ, or anindividual organism, such as a mammal. The mammal can be any suitablemammal, such as a mammal selected from the group consisting of a mouse,rat, guinea pig, hamster, cat, dog, pig, cow, horse, and primate. In oneembodiment, the mammal is a human.

A viral infection is present in a host when a virus replicates itselfwithin the host. A virus contains its own genetic material but uses themachinery of the host to reproduce. The virus may reproduce immediately,whereby the resulting virions destroy a host cell to attack additionalcells. This process is the viral lytic cycle. Alternatively, a virus mayestablish a quiescent infection in a host cell, lying dormant untilenvironmental stimuli trigger re-entry into the lytic replication cycle.Such re-emergence or re-entry into the lytic replication cycle is termedreactivation.

The viral infection may be due to a herpes viral infection. Theherpesvirus may be, e.g., herpes simplex virus (HSV) type 1, herpessimplex virus type 2, varicella zoster virus (VZV), such as VZV-1, orcytomegalovirus. The viral infection may be due to an adenovirusinfection. The adenovirus may be any adenovirus, including, e.g., Types1-5. One embodiment is where the adenovirus is Type 5.

Herpes simplex virus type 1 (HSV-1) and type 2 (HSV-2) are commoninfections worldwide. HSV-2 is the cause of most genital herpes and isgenerally sexually transmitted. In contrast, HSV-1 is usuallytransmitted via nonsexual contacts. Preexisting HSV-1 antibodies canalleviate clinical manifestations of subsequently acquired HSV-2.Furthermore, HSV-1 has become an important cause of genital herpes insome developed countries. Varicella Zoster virus characteristicallyproduces vesicular pruritic disseminated lesions at varying degrees ofmaturity. It occurs most frequently in children, with prodromal malaise,pharyngitis and rhinitis, usually with fever and pruritus (chickenpox).Varicella Zoster virus may cause more severe illness in adults, wherethe lesions are localized and painful, and often involve the trunk(shingles).

Another embodiment of the invention provides a method of preventing ortreating a viral infection in a mammal that has undergone, isundergoing, or will undergo an organ or tissue transplant, comprisingadministering to the mammal an effective amount of an inhibitor of theprotein LSD1 and/or a monoamine oxidase inhibitor before, during, and/orafter the organ or tissue transplant. A non-limiting example would be toadminister an effective amount of an inhibitor of the protein LSD1and/or a monoamine oxidase inhibitor to a mammal receiving (or that hasreceived) an organ or tissue known or suspected to be infected withvirus. Therefore, should the organ or tissue comprise virus in thelatent state, the mammal may still have the transplant, allowing themammal to receive a potentially life-saving transplant while not havingto destroy the organ or tissue.

Another embodiment of the invention provides a method of preventing ortreating a viral infection in a mammal that has undergone or willundergo an organ or tissue transplant, comprising administering to themammal an effective amount of an inhibitor of the protein LSD1 and/or amonoamine oxidase inhibitor before, during, and/or after the organ ortissue transplant, wherein the administration of the inhibitor(s)prevents or treats the viral infection.

Any suitable monoamine oxidase inhibitor (MAOI) can be used in theinventive methods. Examples of MAOIs include pargyline, phenelzine,tranylcypromine, isocarboxazid, moclobemide, selegiline, nialamide, andtoloxatone. In some embodiments of the invention, the MAOI can betranylcypromine, pargyline, phenelzine, isocarboxazid, or selegiline.

Other inhibitors of LSD1 can be used. A suitable inhibitor includes anucleic acid (e.g., RNA), protein, small molecule, or antibody thatspecifically binds to LSD1, inhibits translation of LSD1, inhibitstranscription of LSD1, or otherwise interferes with the biologicalexpression and/or activity of LSD1. One such inhibitor is an RNAinterference (RNAi) inhibitor. The RNAi inhibitor may comprise any RNAsequence that is complementary to the target LSD1 nucleic acid or aportion thereof. Antibodies and RNAi inhibitors of LSD1 can be preparedusing routine techniques. Furthermore, suitable inhibitors can bedetermined using routine techniques, such as employing the EpiQuikHistone Demethylase LSD1 Inhibitor Screening Assay Kit (Epigentek Group,Brooklyn, N.Y.) or the LSD1 Inhibitor Screening Assay Kit (CaymanChemical Company, Ann Arbor, Mich.).

Without intending to be bound by any theory, HCF-1 is a cellulartranscriptional coactivator that is required for the expression of theimmediate early genes (IE), such as the IE genes of α-herpesvirusesHSV-1 and VZV-1 during the initiation of lytic infection. Viruses, suchas HSV and VZV, utilize virion-encapsidated transcriptional activatorsto recruit the HCF-1-Set/MLL1 histone methyl-transferase (HMT) complexesto the viral IE promoters, resulting in histone H3-lysine 4 (H3K4)trimethylation and initiation of IE gene transcription. Furthermore,depletion of HCF-1 results in an increase in the levels of repressivehistone H3-lysine 9 (H3K9) methylation, providing a central role forHCF-1 in modulating chromatin modifications that determine viral geneexpression. A description of the role of HCF-1 in reactivation fromlatency is set forth in Whitlow et al. (J. Virol., 2009; Epub0:JV1.01115-09v1).

LSD1 (also known as BHC110) interacts with HCF-1 and has been shown topossess H3K9 demethylase activity which is important for the activationof nuclear hormone receptor-dependent transcription, cell fatedetermination, and cell cycle progression. LSD1 demethylates lysineresidues via a flavin-adenine-dinucleotide-dependent reaction that isinhibited by MAOIs.

An inhibitor of LSD1 and/or a MAOI can be administered in a composition(e.g., pharmaceutical composition) that can comprise at least onecarrier (e.g., a pharmaceutically acceptable carrier), as well as othertherapeutic agents (e.g., other inhibitors of LSD1 and/or other MAOIs).The composition can be administered by any suitable route, includingparenteral, topical, oral, or local administration. One embodiment ofthe invention is topical administration of an inhibitor of LSD1 and/or aMAOI. Such topical administration may be accomplished using a cream orlotion formulation for, e.g., the clearance of cold sores (HSV-1),genital sores (HSV-2), or shingles (VZV).

The pharmaceutically acceptable carrier (or excipient) is preferably onethat is chemically inert to the MAOI and/or inhibitor of LSD1 and onethat has little or no side effects or toxicity under the conditions ofuse. Such pharmaceutically acceptable carriers include, but are notlimited to, water, saline, Cremophor EL (Sigma Chemical Co., St. Louis,Mo.), propylene glycol, polyethylene glycol, alcohol, and combinationsthereof. The choice of carrier will be determined in part by theparticular MAOI and/or inhibitor of LSD1 as well as by the particularmethod used to administer the composition. Accordingly, there is a widevariety of suitable formulations of the composition.

Preservatives may be used in the pharmaceutical composition. Suitablepreservatives may include, for example, methylparaben, propylparaben,sodium benzoate, and benzalkonium chloride. A mixture of two or morepreservatives optionally may be used. The preservative or mixturesthereof are typically present in an amount of about 0.0001% to about 2%by weight of the total composition.

Suitable buffering agents may be used in the pharmaceutical compositionand may include, for example, citric acid, sodium citrate, phosphoricacid, potassium phosphate, and various other acids and salts. A mixtureof two or more buffering agents optionally may be used. The bufferingagent or mixtures thereof are typically present in an amount of about0.001% to about 4% by weight of the total composition.

The following formulations for oral, aerosol, parenteral (e.g.,subcutaneous, intravenous, intraarterial, intramuscular, intradermal,interperitoneal, and intrathecal), and rectal administration are merelyexemplary and are in no way limiting.

Formulations suitable for oral administration can consist of (a) liquidsolutions, such as an effective amount of the compound dissolved indiluents, such as water, saline, or orange juice; (b) capsules, sachets,tablets, lozenges, and troches, each containing a predetermined amountof the active ingredient, as solids or granules; (c) powders; (d)suspensions in an appropriate liquid; and (e) suitable emulsions. Liquidformulations may include diluents, such as water and alcohols, forexample, ethanol, benzyl alcohol, and the polyethylene alcohols, eitherwith or without the addition of a pharmaceutically acceptablesurfactant, suspending agent, or emulsifying agent. Capsule forms can beof the ordinary hard- or soft-shelled gelatin type containing, forexample, surfactants, lubricants, and inert fillers, such as lactose,sucrose, calcium phosphate, and cornstarch. Tablet forms can include oneor more of lactose, sucrose, mannitol, corn starch, potato starch,alginic acid, microcrystalline cellulose, acacia, gelatin, guar gum,colloidal silicon dioxide, croscarmellose sodium, talc, magnesiumstearate, calcium stearate, zinc stearate, stearic acid, and otherexcipients, colorants, diluents, buffering agents, disintegratingagents, moistening agents, preservatives, flavoring agents, andpharmacologically compatible carriers. Lozenge forms can comprise theactive ingredient in a flavor, usually sucrose and acacia or tragacanth,as well as pastilles comprising the active ingredient in an inert base,such as gelatin and glycerin, or sucrose and acacia, emulsions, gels,and the like containing, in addition to the active ingredient, suchcarriers as are known in the art. A component of the formulation mayserve more than one function.

The inhibitors of LSD1 and/or MAOIs, alone or in combination with othersuitable components, can be made into aerosol formulations to beadministered via inhalation. These aerosol formulations can be placedinto pressurized acceptable propellants, such asdichlorodifluoromethane, propane, nitrogen, and the like. They also maybe formulated as pharmaceuticals for non-pressured preparations, such asin a nebulizer or an atomizer.

Formulations suitable for parenteral administration include aqueous andnon-aqueous, isotonic sterile injection solutions, which can containanti-oxidants, buffers, bacteriostats, and solutes that render theformulation isotonic with the blood of the intended recipient, andaqueous and non-aqueous sterile suspensions that can include suspendingagents, solubilizers, thickening agents, stabilizers, and preservatives.The inhibitors of LSD1 and/or MAOIs may be administered in aphysiologically acceptable diluent in a pharmaceutical carrier, such asa sterile liquid or mixture of liquids, including water, saline, aqueousdextrose and related sugar solutions, an alcohol, such as ethanol,isopropanol, or hexadecyl alcohol, glycols, such as propylene glycol orpolyethylene glycol, glycerol ketals, such as2,2-dimethyl-1,3-dioxolane-4-methanol, ethers, such aspoly(ethyleneglycol) 400, an oil, a fatty acid, a fatty acid ester orglyceride, or an acetylated fatty acid glyceride with or without theaddition of a pharmaceutically acceptable surfactant, such as a soap ora detergent, suspending agent, such as pectin, carbomers,methylcellulose, hydroxypropylmethylcellulose, orcarboxymethylcellulose, or emulsifying agents and other pharmaceuticaladjuvants.

Oils, which can be used in parenteral formulations, include petroleum,animal, vegetable, or synthetic oils. Specific examples of oils includepeanut, soybean, sesame, cottonseed, corn, olive, petrolatum, andmineral. Suitable fatty acids for use in parenteral formulations includeoleic acid, stearic acid, and isostearic acid. Ethyl oleate andisopropyl myristate are examples of suitable fatty acid esters.

Suitable soaps for use in parenteral formulations may include fattyalkali metal, ammonium, and triethanolamine salts, and suitabledetergents include (a) cationic detergents such as, for example,dimethyl dialkyl ammonium halides, and alkyl pyridinium halides, (b)anionic detergents such as, for example, alkyl, aryl, and olefinsulfonates, alkyl, olefin, ether, and monoglyceride sulfates, andsulfosuccinates, (c) nonionic detergents such as, for example, fattyamine oxides, fatty acid alkanolamides, andpolyoxyethylene-polypropylene copolymers, (d) amphoteric detergents suchas, for example, alkyl-beta-aminopropionates, and 2-alkyl-imidazolinequaternary ammonium salts, and (3) mixtures thereof.

The parenteral formulations will typically contain from about 0.5 toabout 25% by weight of the active ingredient in solution. Suitablepreservatives and buffers can be used in such formulations. In order tominimize or eliminate irritation at the site of injection, suchcompositions may contain one or more nonionic surfactants having ahydrophile-lipophile balance (HLB) of from about 12 to about 17. Thequantity of surfactant in such formulations ranges from about 5% toabout 15% by weight. Suitable surfactants include polyethylene sorbitanfatty acid esters, such as sorbitan monooleate and the high molecularweight adducts of ethylene oxide with a hydrophobic base, formed by thecondensation of propylene oxide with propylene glycol. The parenteralformulations can be presented in unit-dose or multi-dose sealedcontainers, such as ampoules and vials, and can be stored in afreeze-dried (lyophilized) condition requiring only the addition of thesterile liquid carrier, for example, water, for injections, immediatelyprior to use. Extemporaneous injection solutions and suspensions can beprepared from sterile powders, granules, and tablets.

The inhibitors of LSD1 and/or MAOIs may be administered as an injectableformulation. The requirements for effective pharmaceutical carriers forinjectable compositions are well known to those of ordinary skill in theart. See Pharmaceutics and Pharmacy Practice, J. B. Lippincott Co.,Philadelphia, Pa., Banker and Chalmers, eds., pages 238-250 (1982), andASHP Handbook on Injectable Drugs, Toissel, 4th ed., pages 622-630(1986).

Topical formulations, including those that are useful for transdermaldrug release, are well known to those of skill in the art and aresuitable in the context of embodiments of the invention for applicationto skin.

The concentration of a compound of embodiments of the invention in thepharmaceutical formulations can vary, e.g., from less than about 1%,usually at or at least about 10%, to as much as 20% to 50% or more byweight, and can be selected primarily by fluid volumes, and viscosities,in accordance with the particular mode of administration selected.

Methods for preparing administrable (e.g., parenterally administrable)compositions are known or apparent to those skilled in the art and aredescribed in more detail in, for example, Remington: The Science andPractice of Pharmacy, Lippincott Williams & Wilkins; 21st ed. (2005).

In addition to the aforedescribed pharmaceutical compositions, theinhibitors of LSD1 and/or MAOIs thereof can be formulated as inclusioncomplexes, such as cyclodextrin inclusion complexes, or liposomes.Liposomes can serve to target the MAOI and/or inhibitor of LSD1 to aparticular tissue. Liposomes also can be used to increase the half-lifeof the MAOI and/or inhibitor of LSD1. Many methods are available forpreparing liposomes, as described in, for example, Szoka et al., Ann.Rev. Biophys. Bioeng., 9, 467 (1980) and U.S. Pat. Nos. 4,235,871,4,501,728, 4,837,028, and 5,019,369.

When inhibitors of LSD1 and/or MAOIs are administered with one or moreadditional therapeutic agents, including additional inhibitors of LSD1and/or MAOIs as the additional therapeutic agents, one or moreadditional therapeutic agents can be coadministered to the mammal. By“coadministering” is meant administering one or more additionaltherapeutic agents and the inhibitors of LSD1 and/or MAOIs sufficientlyclose in time such that the inhibitors of LSD1 and/or MAOIs can enhancethe effect of one or more additional therapeutic agents. In this regard,the inhibitors of LSD1 and/or MAOIs can be administered first and theone or more additional therapeutic agents can be administered second, orvice versa. Alternatively, the inhibitors of LSD1 and/or MAOIs and theone or more additional therapeutic agents can be administeredsimultaneously.

The delivery systems useful in the context of embodiments of theinvention may include time-released, delayed release, and sustainedrelease delivery systems such that the delivery of the inventivecomposition occurs prior to, and with sufficient time to cause,sensitization of the site to be treated. The inventive composition canbe used in conjunction with other therapeutic agents or therapies. Suchsystems can avoid repeated administrations of the inventive composition,thereby increasing convenience to the subject and the physician, and maybe particularly suitable for certain composition embodiments of theinvention.

Many types of release delivery systems are available and known to thoseof ordinary skill in the art. They include polymer base systems such aspoly(lactide-glycolide), copolyoxalates, polycaprolactones,polyesteramides, polyorthoesters, polyhydroxybutyric acid, andpolyanhydrides. Microcapsules of the foregoing polymers containing drugsare described in, for example, U.S. Pat. No. 5,075,109. Delivery systemsalso include non-polymer systems that are lipids including sterols suchas cholesterol, cholesterol esters, and fatty acids or neutral fats suchas mono-di- and tri-glycerides; hydrogel release systems; sylasticsystems; peptide based systems; wax coatings; compressed tablets usingconventional binders and excipients; partially fused implants; and thelike. Specific examples include, but are not limited to: (a) erosionalsystems in which the active composition is contained in a form within amatrix such as those described in U.S. Pat. Nos. 4,452,775, 4,667,014,4,748,034, and 5,239,660 and (b) diffusional systems in which an activecomponent permeates at a controlled rate from a polymer such asdescribed in U.S. Pat. Nos. 3,832,253 and 3,854,480. In addition,pump-based hardware delivery systems can be used, some of which areadapted for implantation.

The terms “treat,” “prevent,” and “inhibit” as well as words stemmingtherefrom, as used herein, do not necessarily imply 100% or completetreatment, prevention, or inhibition. Rather, there are varying degreesof treatment, prevention, or inhibition of which one of ordinary skillin the art recognizes as having a potential benefit or therapeuticeffect. In this respect, the inventive methods can provide any amount ofany level of treatment, prevention, or inhibition of a conditionassociated with, e.g., LSD1 activity, such as demethylation of histones,in a host or mammal. Furthermore, the treatment, prevention, orinhibition provided by the inventive methods can include treatment,prevention, or inhibition of one or more conditions or symptoms of thedisease being treated, prevented, or inhibited. Also, for purposesherein, “prevention” or “inhibiting” can encompass delaying the onset ofthe disease or a symptom or condition thereof.

An “effective amount” refers to a dose that is adequate to prevent,treat, or inhibit a condition associated with, e.g., LSD1 activity.Amounts effective for a therapeutic or prophylactic use will depend on,for example, the stage and severity of the disease or disorder beingtreated, the age, weight, and general state of health of the patient,and the judgment of the prescribing physician. The size of the dose willalso be determined by the compound selected, method of administration,timing and frequency of administration as well as the existence, nature,and extent of any adverse side-effects that might accompany theadministration of a particular compound and the desired physiologicaleffect. For example, the dose of the inhibitor to be administered can beabout 0.1 mg to 10 g per day (e.g., 0.5 mg, 1 mg, 5 mg, 10 mg, 20 mg, 30mg, 40 mg, 50 mg, 60 mg, 70 mg, 80 mg, 90 mg, 100 mg, 150 mg, 200 mg,250 mg, 300 mg, 350 mg, 400 mg, 450 mg, 500 mg, 550 mg, 600 mg, 650 mg,700 mg, 750 mg, 800 mg, 850 mg, 900 mg, 950 mg, 1000 mg, 2 g, 3 g, 4 g,5 g, 6 g, 7 g, 8 g, 9 g, or ranges of any of the values describedherein). Alternatively, the dose of inhibitor to be administered can be0.001 mg/kg to 200 mg/kg per day (e.g., 0.01 mg/kg, 0.05 mg/kg, 0.1mg/kg, 0.5 mg/kg, 1 mg/kg, 5 mg/kg, 10 mg/kg, 50 mg/kg, 100 mg/kg, 150mg/kg, or ranges of any of the values described herein). It will beappreciated by one of skill in the art that various diseases ordisorders could require prolonged treatment involving multipleadministrations, perhaps using inhibitors of LSD1 and/or monoamineoxidase inhibitors in each or various rounds of administration.

A number of transfection techniques are generally known in the art (see,e.g., Graham et al., Virology, 52: 456-467 (1973); Sambrook et al.,Molecular Cloning: A Laboratory Manual, 3rd ed., Cold Spring HarborPress, Cold Spring Harbor, N.Y. 2001; Davis et al., Basic Methods inMolecular Biology, Elsevier (1986); and Chu et al., Gene, 13: 97 (1981).Transfection methods include calcium phosphate co precipitation (see,e.g., Graham et al., supra), direct micro injection into cultured cells(see, e.g., Capecchi, Cell, 22: 479-488 (1980)), electroporation (see,e.g., Shigekawa et al., BioTechniques, 6: 742-751 (1988)), liposomemediated gene transfer (see, e.g., Mannino et al., BioTechniques, 6:682-690 (1988)), lipid mediated transduction (see, e.g., Felgner et al.,Proc. Natl. Acad. Sci. USA, 84: 7413-7417 (1987)), and nucleic aciddelivery using high velocity microprojectiles (see, e.g., Klein et al.,Nature, 327: 70-73 (1987)).

The following examples further illustrate the invention but, of course,should not be construed as in any way limiting its scope.

EXAMPLES

Cell culture and viral infections: HeLa, BS-C-1, CV-1, Vero, MeWo, VZV(Ellen), and adenovirus type 5 stocks were obtained from American TypeCulture Collection (ATCC). HSV-1 strain 17 was provided by N. Fraser(University of Pennsylvania School of Medicine). HSV (strain 17)infections were done by exposing uninfected cells to HSV infected celllysates in DMEM medium containing 1% fetal bovine serum. At 1 hr postinfection, the medium was replaced with DMEM containing 10% serum andincubation was continued. VZV infections were done by directlyoverlaying VZV infected BS-C-1 cells onto naïve cells. MCF7 inducibleLSD1 shRNA cells were provided by X. Chen (University of California atDavis).

Antibodies: IE62 (Narayanan et al., Proc. Natl. Acad. Sci. USA, 2007,104, 10835-40); HCF-1 (Kristie et al., J. Biol. Chem., 1995, 270,4387-94); Normal rabbit IgG, H3K4-trimethyl, H3K9-monomethyl,H3K9-dimethyl, HP1γ, CoREST (Millipore/Upstate Biotechnologies, LakePlacid, N.Y.: 12-370, 05-745, 07-450, 05-690, 07-455, respectively);FLAG-M2, V5 (Sigma-Aldrich), HA (Roche), Tubulin, TBP, Sp1 (Santa CruzBiotechnologies, Santa Cruz, Calif.: SC-1904, SC-273, SC-59,respectively); MLL1, Set1, RbBP5, BRAF35 (Bethyl Laboratories,Montgomery, Tex.: BL1408/1289, BL1193, BL766, A301-097A.1,respectively); Histone H3, LSD1, H3K9-trimethyl, HDAC1, H3K9-monomethyl,H3K9-dimethyl (Abcam, Cambridge, Mass.: ab1791,ab17721-ChIP/ab37165-Western, ab8898, ab7028, ab9045, ab1220,respectively); ICP0, ICP27, ICP4 (Goodwin Institute, Plantation, Fla.:GICR1112, GICR1113, and GICR1101, respectively); ICP8 (provided byWilliam Ruyechan, University at Buffalo, SUNY), and Neurofilament 200(Sigma N012).

Example 1

This example demonstrates the role of histone methylation in herpesvirusgene expression.

Methods

Reporter assays: The VZV model IE promoter-reporter (pIE62P-61), IE62expression plasmid (pCMV-IE62), and luciferase reporter assays have beendescribed previously (Narayanan et al., Proc. Natl. Acad. Sci. USA,2007, 104, 10835-40). The VZV model IE promoter-luciferase reportercontained the minimal promoter sequences required for IE62 mediatedinduction (−61 to +73 relative to the IE62 transcription initiationsite) in pGL3Basic (pIE62P-61). pCMV-IE62 expresses the IE62 activatorunder control of the CMV IE promoter (Narayanan et al. J. Biol. Chem.,2005, 280, 1369-75). The HSV IE promoter-luciferase reporter containedthe promoter sequences for VP 16 mediated induction (−171 to +57relative to the ICP0 transcription initiation site) in pGL4.18(pICP0-171). Luciferase reporter activity was measured and analyzed 24hrs later as described using the Dual-Luciferase Assay Kit (Promega,Madison, Wis.) in a Berthold luminometer (Narayanan et al., Proc. Natl.Acad. Sci. USA, 2007, 104, 10835-40). All activity units were normalizedby protein concentration and the activity of the internal controlvector.

Chromatin immunoprecipitations (ChIP) from extracts of control, HCF-1depleted, and LSD1 depleted cells were done essentially as described(Narayanan et al., Proc. Natl. Acad. Sci. USA, 2007, 104, 10835-40).Recovered DNA was subjected to semi-quantitative PCR with dilutions ofinput DNA to insure linearity using PCR Supermix (Invitrogen, Carlsbad,Calif.) with the following conditions: 95° C. for 2 min followed by 20cycles of 95° C. for 30 sec; 59° C. for 30 sec; 72° C. for 30 sec. Thesignal intensities of individual bands, resolved in ethidium bromideagarose gels, were calculated as a percentage of the intensity of theinput extract after subtraction of the appropriate background antibodycontrols. Signal intensities of resolved bands were quantitated using aKodak 4000 MM Image Station.

Samples also were analyzed, in triplicate, by qPCR using ABI Sybr GreenPCR Master Mix. For qPCR, samples were run on a ABI PRISM 7900HT withthe following conditions: 95° C. for 10 min followed by 40 cycles of 95°C. for 15 sec; 57° C. for 15 sec; 72° C. for 30 sec. Results wereanalyzed using ABI SDS 2.3 software.

Primer sets for ChIP were as follows:

VZV IE model promoter 5′R ACTAGCAAAATAGGCTGTCCCCAG (SEQ ID NO: 1)3′R CCTTTCTTTATGTTTTTGGCGTC (SEQ ID NO: 2) VZV IE62 promoter (genomic)5′P GAAATAGACACCGGGCGTACATC (SEQ ID NO: 3) 3′P GAATTTAGACGTACCCGAGTTTTCC(SEQ ID NO: 4) VZV IE62 coding (genomic) 5′C GTTGCAGACGATCATGTGGTTTC(SEQ ID NO: 5) 3′C GTCGCGAGGGTGCTCTCG (SEQ ID NO: 6)HSV ICP0 promoter (distal) P1-5′ CGCGGGTCGCTCAATGAAC (SEQ ID NO: 7)P1-3′ GCCCGGCCCCCGATT (SEQ ID NO: 8) HSV ICP0 promoter (proximal) P15-5′CCCTGGCCCGACAGTCTG (SEQ ID NO: 9) P15-3′ CAGGCCGGCGGGTACTC(SEQ ID NO: 10) GAPDH-pr GPr-5′ CGGACTGCAGCCCTCCC (SEQ ID NO: 23) GPr-3′CCTTCCCAGTTTCCGACTGTCC (SEQ ID NO: 24) Actin-prAPr-F TGGCTCAGCTTTTTGGATTC (SEQ ID NO: 21) APr-R GGGAGGATTGGAGAAGCAGT(SEQ ID NO: 22)

Results

A reporter system in which HeLa cells were transfected with a model VZVIE promoter-reporter was utilized. The state of histone H3K4 and H3K9methylation was assessed by chromatin immunoprecipitation/qPCR assays inthe presence and absence of the VZV IE activator (IE62). In the absenceof the activator, repressive H3K9 methylation accumulated on thepromoter. In the presence of the activator, H3K9 methylation was reducedand positive H3K4 trimethylation was enhanced.

These data suggest that, in addition to the Set1/MLL1 H3K4methyl-transferase, an H3K9 demethylase is likely required in order toremove these repressive marks for activation of transcription.

Example 2

This example demonstrates the roles of HCF-1, Set-1 and LSD1 in viral IEgene transcription.

Methods

The methods of Example 1 were followed. In addition, for LSD1 depletion,4×10⁴ cells were transfected with 1 μg control RNAi, LSD-1, or LSD-2shRNA constructs (Origene TR20003, TI365146, and TI365147, respectively)using Fugene 6 according to the manufacturer's recommendations (RocheApplied Science, Indianapolis, Ind.). 48 hours post transfection, thecells were cotransfected with 100 ng reporter construct, 0.1 ng RL-CMVinternal control, and increasing amounts of pCMV-IE62. Luciferasereporter activity was measured and analyzed 24 hours later as describedin Example 1.

Results

In concert with HCF-1 and Set1, LSD1 also was recruited to the model IEpromoter. Reduction in the levels of LSD1 using two distinctLSD1-RNAi(s) resulted in reduced induction of the IE reporter,demonstrating that LSD1 was important for IE62 mediated transcriptionalactivation. In a similar manner, depletion of LSD1 reduced the inductionof the induction of an HSV-1 IE reporter. Transfection of a constructexpressing LSD1 stimulated the reporter, while that expressing an LSD1mutant had no significant impact.

Example 3

This example demonstrates the recruitment of LSD1 is dependent upon thecoactivator HCF-1.

Methods

The methods of Example 1 were followed. In addition, HSV (strain 17)infections were done by exposing uninfected cells to HSV infected celllysates in DMEM medium containing 1% fetal bovine serum. At 1 hr postinfection, the medium was replaced with DMEM containing 10% serum andincubation was continued. VZV infections were done by directlyoverlaying VZV-infected MeWo cells unto naïve BS-C-1 cells and thenusing these infected BS-C-1 cells to overlay new BS-C-1 naïve cells.Depletion of HCF-1 was accomplished using anti-HCF-1 RNAi constructs.For HCF-1 depletion, HCF-1 RNAi was used as described in Narayanan etal., J. Biol. Chem., 2006, 280, 1369-1375.

Results

In HCF-1 depleted cells, H3K4 trimethylation of the model promoter wasreduced (7-fold) while H3K9 methylation was enhanced 8-9-fold,correlating with a decrease in the promoter occupancy by Set1 and LSD1.In contrast, occupancy of the DNA binding viral IE activator (IE62) isHCF-1 independent in this system and was equivalent in both HCF-1(−) andHCF-1(+) cells.

Cells were infected with VZV and the chromatin modification status andprotein occupancy was assessed for the viral IE promoters in HCF-1depleted and control cells. In these experiments, HCF-1 was onlypartially depleted (52%) to prevent impacts on cell cycle progressionand changes in the levels of the viral IE62 activator. In control cells,promoter occupancy by IE62, HCF-1, Set1, MLL1, and LSD1 were allsubstantial, in contrast to occupancy of control coding sequences.Binding of these proteins correlated with high levels of H3K4trimethylation and near background levels of repressive H3K9methylation. Equivalent results were also obtained in analysis of thepromoters of the HSV IE genes. However, in cells partially depleted forHCF-1, occupancy by Set1 and LSD1 decreased with a correlating decreasein the levels of H3K4 trimethylation and increase in the levels of therepressive H3K9 methylation.

These data indicate that the HCF-1 dependent recruitment of Set1 andLSD1 was important to remove the repressive H3K9 chromatin marks fromthe viral promoter and promote H3K4 methylation.

Example 4

This example demonstrates the significance of the recruitment of LSD1 toenable productive VZV infection.

Methods

Viral infections were performed as described in Example 3. Depletion ofLSD1 was accomplished using a MCF7 doxycycline inducible LSD1-RNAi cellline. Expression of the LSD1 RNAi was induced for 96 hours prior toinfection of the depleted cells with VZV.

qRT-PCR: Oligo dT primed cDNA was produced from total RNA usingRNAqueous-4PCR and RETROscript (Ambion, Austin, Tex.) according to themanufacturer's recommendations. cDNA was quantitated by qPCR.

Primer sets for RT-PCR were as follows:

ICP0 F CCCACTATCAGGTACACCAGCTT (SEQ ID NO: 11) R CTGCGCTGCGACACCTT(SEQ ID NO: 12) 1E62 F TGGACGAGGCGGCACATAG (SEQ ID NO: 13)R AGGGCGTGGCGGCAAAACAC (SEQ ID NO: 14) ICP27 F GCATCCTTCGTGTTTGTCATTCTG(SEQ ID NO: 15) R GCATCTTCTCTCCGACCCCG (SEQ ID NO: 16) s15F TTCCGCAAGTTCACCTACC (SEQ ID NO: 17) R CGGGCCGGCCATGCTTTACG(SEQ ID NO: 18) ICP4 F TGCTGCTGCTGTCCACGC (SEQ ID NO: 19)R CGGTGTTGACCACGATGAGCC (SEQ ID NO: 20) Actin F TGGCTCAGCTTTTTGGATTC(SEQ ID NO: 21) R GGGAGGATTGGAGAAGCAGT (SEQ ID NO: 22) Sp1F TCAGAACCCACAAGCCCAAAC (SEQ ID NO: 25) R TGCCAGCAGGAATGGAAGC(SEQ ID NO: 26) TBP F TGACCCCCATCACTCCTGC (SEQ ID NOT: 27)R CGTGGTTCGTGGCTCTCTTATC (SEQ ID NO: 28)

For the detection of viral IE and cellular mRNAs post explant, 8 gangliafrom HSV latently infected or mock infected Balb/c mice were explantedin the presence of DMSO, 100 μM acyclovir, or 2 mM TCP for the indicatedtimes. cDNAs were prepared from total RNA and amplified by qPCR(cellular Sp1 and TBP controls) or nested PCR (viral IE). Primer setsfor the nested RT-PCR were as follows:

ICP4 Primary F GCGAGCAGCCCCAGAAACTC (SEQ ID NO: 29)R ACGACGATAACCCCCACCC (SEQ ID NO: 30) ICP4 Secondary/NestedF GGACAGCAGCAGCACGCC (SEQ ID NO: 31) R ATCCCCGACCCCGAGGACG(SEQ ID NO: 32) ICP27 Primary F CCCCAGGACCCCATTATCG (SEQ ID NO: 33)R TTCTCTCCGACCCCGACACCAAGG (SEQ ID NO: 34) ICP27 Secondary/NestedF GCTGGATAACCTCGCCACG (SEQ ID NO: 35) R CAGAATGACAAACACGAAGGATGC(SEQ ID NO: 36)

cDNA samples were normalized to one another according to the level ofcellular control Sp1 mRNA. To insure linearity and sensitivity of nestedPCR reactions, samples were amplified in parallel with dilutions of cDNAprepared from 3T3 cells infected with HSV at 6.4×10⁻⁵ pfu/cell for 4hrs. PCR products were resolved in agarose gels and quantitated usingKodak Image Station 4000 MM Digital Imaging System.

Communoprecipitations: 2.5×10⁶ HEK293 cells were transfected with 9.6 ugpHA-LSD and 14.4 ug pHCF-FLAG or pHCF-V5 expression plasmids usingLipofectamine 2000 (Invitrogen, Carlsbad, Calif.). Forty-eight hourslater, nuclear extracts were incubated with FLAG-M2 agarose beads(Sigma, St. Louis, Mo.) at 4° C. for 1 hr in NP-40 buffer (50 mM Tris pH7.5, 150 mM NaCl, 0.1% NP-40, 5% glycerol, 1 mM NaF, 10 mMβ-glycerophosphate, 0.1 mM Na₃VO₄, Complete protease inhibitor). Forendogenous coimmunoprecipitations, antibodies were prebound to Protein GDynabeads and incubated with HeLa cell nuclear extracts in NP-40 bufferovernight. Immunoprecipitates were washed 5 times with binding buffer,eluted in SDS sample buffer, and resolved in 4-20% Tris-Glycine gels.Western Blots of resolved extracts and immunoprecipitates were developedusing Pierce SuperSignal West Dura.

Results

The levels of LSD1 and VZV IE protein and mRNAs were determined byWestern blot and qRT-PCR, respectively. Depletion of 60% of the cellularLSD1 reduced the levels of the viral IE protein by 66% and mRNA by 78%.Similar impacts on the expression of the HSV IE proteins also were seenin LSD1 depletion experiments and significant levels of viral IE geneexpression was recovered in LSD1 depleted cells by exogenous expressionof wild-type LSD1, but not by expression of a LSD1 catalytic mutant or amutant lacking the amine oxidase domain. Depletion of LSD1 resulted inan increased accumulation of nucleosomes bearing repressive H3K9methylation on the viral IE promoters.

To determine whether recruitment of LSD1 was based on an interactionwith HCF-1, immunoprecipitates of endogenous LSD1 were probed for HCF-1.Immunoprecipitation with either Set1- or LSD1-specific antibodiesresulted in efficient coimmunoprecipitation of HCF-1.

As HCF-1 is a component of the Set1 and MLL1 histone H3-lysine 4methyl-transferase complexes (HMTs), whether LSD1 was associated withthe HCF-1 HMT complex or was present in a distinct HCF-1 complex wasdetermined by probing HCF-1 and LSD1 immunoprecipitates for RbBP5, acommon core subunit of the Set1 and MLL HMT complexes. Both proteinscoimmunoprecipitated the HMT core subunit, suggesting that LSD1 wasassociated with the HCF-1 HMT complex.

To further characterize the HCF-1/LSD1 complex, immunoprecipitates fromcells expressing epitope tagged HCF-1 and LSD1 were probed for cofactorsassociated with the repressive CoREST/LSD1 complex. Such factors werepresent in the LSD1 immunoprecipitate but were absent from theactivating HCF-1/LSD1 complex.

Based on these data, HCF-1 couples the demethylase LSD1 with theSet1/MLL1 methyltransferase complex, providing both specificitiesrequired to activate the expression of transcriptionally repressed genes(IE gene transcription).

To further define the requirement of HCF-1 in viral reactivation,chromatin immunoprecipitation assays were used to demonstrate that HCF-1is rapidly recruited to the IE promoter-enhancer domains of latent viralgenomes (e.g., HSV-1) during the initial stages of reactivation. Insensory neurons, recruitment of HCF-1 was detected as early as 1 hr postinduction of reactivation and occupancy increased by 4 hrs postinduction. This time course correlates well with the nuclear transportof HCF-1, where the number of neurons exhibiting nuclear localizationincreases over the course of 6 hrs post stimulation. Additionally, HCF-1occupancy of viral IE promoter domains correlated with the occupancy byRNAPII and accumulation of viral IE mRNAs. The recruitment of HCF-1 iscorrelated with the association of RNAPII and the detection of IE mRNA.

These data support a model in which HCF-1 is involved in both theinitiation of lytic infection and the reactivation of HSV-1 fromlatency.

Example 5

This example demonstrates MAOI inhibition of LSD1 and the α-herpesviruslytic cycle.

Methods

For treatment with MAOIs, cells were pretreated with the indicatedconcentration of drug for 4-5 hrs prior to infection and maintainedthroughout infection. Pargyline (p-8013) and Tranylcypromine (p-8511)were obtained from Sigma (St. Louis, Mo.).

Cell infections: For HSV, cells were infected with 0.1 plaque formingunits (PFU) of HSV per cell for 24 hrs in the presence of 2 mMTranylcypromine (TCP) or control DMSO. The resulting viral yields weredetermined by titer of infected cell lysates on Vero cell monolayers.VZV infections were performed as in Example 3.

Results

The impact of the MAOIs Pargyline and TCP on VZV and HSV infection wasinvestigated. In each case, treatment with the inhibitor resulted in adose dependent decrease in viral mRNA and IE proteins (FIGS. 1 and 2)with no impact on cellular protein controls and no significant cellulartoxicity. In addition, viral yields from cells infected with HSV in thepresence of TCP were reduced nearly 1000-fold. These results suggestedthat inhibition of LSD1 demethylase activity with MAOIs results in theaccumulation of repressive chromatin on the viral IE promoters, similarto that observed in LSD1 RNAi-mediated depletions (see Example 2) In thepresence of TCP or Pargyline, nucleosomes bearing repressive marksaccumulated on the viral IE promoters as detected by the increase inhistone H3 and H3K9 methylation. These results support a model wherebyLSD1 is required to prevent accumulation of repressive H3K9 methylation,thereby allowing initiation of productive infection by bothα-herpesviruses.

In the presence of TCP, a high level of nucleosomes bearing repressivemarks accumulated on the viral IE promoters as detected by thesubstantial increase in histone H3 and H3K9 trimethylation. In contrast,the level of H3K4 trimethylation did not significantly change, eventhough the level of assembled chromatin increased. It is important tonote, that even in untreated cells, repressive H3K9 methylation isdetected as early as 30 minutes post infection and these marks decreasedover time as HCF-1 dependent complexes were recruited to the viral IEpromoters.

In addition to the increase in mono- and di-methyl H3K9, inhibition ofLSD1 ultimately resulted in an increase in the level of repressiveH3K9-trimethylation and promoter occupancy by the heterochromatinprotein 1 (HP1). As LSD1 only removes mono- and di-methyl modifications,the increase in H3K9 trimethyl marks in the absence of LSD1 couldreflect the increased levels of dimethyl substrates that accumulateduring chromatin assembly on the viral genome. Even in the presence ofLSD1, H3K9 trimethyl marks are readily detected on the viral IEpromoters during the early stages of infection, suggesting thatadditional H3K9 demethylase(s) of the Jmjd family could be required inconjunction with LSD1 to promote viral IE gene expression. Therequirement for LSD1 to promote viral IE expression identifies it as atarget for inhibition of α-herpesviruses lytic infection.

These data show that MAOIs inhibit LSD1 and the α-herpesvirus lyticcycle.

Example 6

This example demonstrates that MAOIs used to inhibit LSD1 and theα-herpesvirus lytic cycle also inhibit the α-herpesvirus re-activationcycle.

Methods

Latently infected mice and trigeminal ganglia: Balb/c mice were infectedwith 5×10⁵ PFU HSV-1 (strain 17) per eye after corneal scarification.Latently infected mice were sacrificed 30 days post clearance of theprimary infection and trigeminal ganglia were rapidly explanted intoculture in the presence or absence of TCP or control (DMSO oracyclovir). Post explant incubation as indicated, the ganglia werehomogenized and briefly sonicated. The reactivated viral yield of eachganglia was determined by titering the clarified supernatant on Verocells.

Statistical analyses: Statistical comparisons were made using Wilcoxonsigned rank test (paired ganglia) with a statistical signficance of<0.05; Kruskal-Wallis test with post hoc Dunn's multiple comparison test(drug titration and reversal) with a statistical significance of <0.025;Mann-Whitney U test with Dunn's post hoc adjustment (non-paired gangliatime course) with a statistical significance of <0.025; or Fischer'sExact Test with a statistical significance of <0.05. Analyses were madeusing Prism (V5.0a) and are expressed as the mean+/−s.e.m.

Non-paired ganglia time course statistic analysis: Comparisons were madebetween control (DMSO) and TCP treated ganglia on day 2 and day 4 postexplant. Each comparison utilized a one-sided Mann-Whitney U testfollowed by application of the Dunn's post hoc adjustment to thep-values. Significant differences were found between DMSO and TCP on day2 (p=0.0043) and on day 4 (p=0.0011). Both of these differences arestill significant after applying the Dunn's adjustment as both are lessthan the alpha level (α=0.025). The Mann-Whitney tests used an exactp-value for small sample sizes. n=6 for each sample set.

Paired ganglia statistical analysis: Each data point was the result of asingle ganglia divided and treated in the presence and absence of TCP.Therefore, a Wilcoxon signed rank test was used to assess differencesbetween each treated and untreated sample. The significant differencewas p=0.0002. This test used an exact p-value for small sample sizeswith an α level of 0.05. n=16 for each sample set.

TCP titration statistical analysis: To determine at what concentrationTCP that would produce a significant reduction in viral load, aKruskal-Wallis (K-W) test with Dunn's post hoc comparison was used. TheK-W test was appropriate due to the comparison of five samples of datathat are not normally distributed. Significant differences were foundbetween the five groups (p=0.0007, K-W=19.39). To determine which groupsare significantly different, a Dunn's post hoc comparison was done.Assessment of each concentration of TCP as compared to the control(DMSO) demonstrated that 1.0, 1.5, and 2.0 mM data sets showedsignificant differences (p<0.05) while 0.5 mM did not. Comparing eachconcentration of TCP against the next higher concentration demonstratedthat 1.0 mM was significantly better at repressing viral yields than 0.5mM. No significant difference was found between 1.0 and 1.5 or 1.5 and2.0 mM data sets. n=5 for control, 0.5, 1.0, and 2.0 mM; n=6 for 1.0 mM.

TCP reversal statistical analysis: Viral yields from ganglia treatedwithout TCP for 2 days, treated with TCP for 2 days, or treated with TCPfor 2 days followed by incubation in the absence of drug for 3 days werecompared using the K-W test followed by Dunn's post hoc comparisons. TheK-W test was appropriate due to the comparison of three samples of datathat are not normally distributed. Significant differences were foundbetween the groups (p=0.0007, K-W=10.3). A significant difference wasdemonstrated between the TCP and TCP-R data sets using Dunn's post hoccomparison (α=0.025). n=4 for TCP; n=6 for TCP-R.

Immunofluoresence statistical analysis: Trigeminal ganglia (16DMSO/Control, 15 acyclovir, and 15 TCP) were fixed at 48 hours postexplants. Serial sections representing the entire ganglia were stainedand scored by confocal microscopy. Ganglia were scored as HSV+ when anysection exhibited ICP8 specific staining. Of the 15 TCP treated ganglia,only one section of one ganglia exhibited a single ICP8+ neuron. AFischer's Exact Test demonstrated that there was significant differencebetween DMSO, acyclovir, and TCP treated ganglia (p=0.00002). The OddsRatio indicated that the odds of viral reactivation were 1.4444 timeshigher for the DMSO treated samples as compared to the acyclovir treatedsamples, 60.6667 times higher for the DMSO treated samples as comparedto the TCP treated samples, and 42.0000 times higher for the acyclovirtreated samples as compared to the TCP treated samples.

Results

In addition to the lytic cycle, α-herpesviruses establish persistentlatent infections and cycles of reactivation in sensory neurons that arecharacterized by alterations in chromatin modifications on the viral IEpromoters. HSV latently infected mice were induced to reactivate virusby tissue explant of the trigeminal sensory ganglia (TGs) for 2 or 4days in the presence and absence of the MAOI TCP (FIG. 3). Strikingly,TCP significantly reduced the reactivation of HSV-1 as compared to thecontrol treated ganglia (p values=0.0043 and 0.0011 for days 2 and 4,respectively). Similar results were observed for the MAOI selegiline(data not shown).

Due to the potential variance in the viral load of individual animals,these studies were repeated using a direct paired analysis in which eachhalf of a latently infected ganglia was explanted in the presence andabsence of TCP (FIG. 4). These results confirmed the significance of TCPinhibition of viral reactivation (p value=0.0002). While the level ofTCP used in these tissue explant experiments was equivalent to thatdetermined to inhibit LSD1 in cell culture, titration of the drugsuggested that lower levels also were effective (FIG. 5). To rule outthe impacts of potential TCP toxicity, latently infected ganglia wereexplanted in the presence of TCP for 2 days followed by incubation inthe absence of drug for 3 days. The high level of viral reactivationfollowing drug removal indicated that toxicity was not responsible forthe suppression of viral reactivation (FIG. 6).

These viral yield studies suggested that inhibition of LSD1 with MAOIsprevented the initiation of viral reactivation in the sensory neurons.However, it remained formally possible that TCP inhibited lytic spreadof the virus in the ganglia but not the initial reactivation events.Therefore, latently infected ganglia were explanted in the presence ofDMSO (control vector), acyclovir (to prevent viral DNA replication andspread), or TCP for 48 hrs. Ganglia were fixed and sections were probedwith antibodies to HSV ICP8, the viral-encoded single stranded bindingprotein. In ganglia explanted in DMSO, clusters of ICP8 (+) neurons weredetected throughout multiple sections in 13 of 16 ganglia, representinginitiating neurons as well as infected neurons and support cellsresulting from lytic spread in the ganglia. In the presence ofacyclovir, distinct ICP8 (+) neurons were clearly detected in severalsections in 9 of 12 ganglia, representing the primary neurons undergoingviral reactivation. Strikingly, in the presence of TCP, only a singleICP8 (+) neuron was detected in 1 of 15 ganglia explanted, clearlydemonstrating that TCP inhibited viral reactivation rather thaninhibiting lytic spread of the infection through the ganglia (pvalue=0.00002).

As an additional approach to demonstrating TCP inhibition of HSVreactivation, cDNA was prepared from RNA isolated from paired latentlyinfected ganglia explanted in the presence of acyclovir or TCP for 12hrs and analyzed by nested PCR for the detection of a representative HSVIE mRNA (ICP27). In the presence of control acyclovir, ICP27 mRNA wasreadily detected at a level comparable to that in control cDNA producedfrom 3T3 cells infected with HSV. In contrast, in ganglia explanted inthe presence of TCP, the level of ICP27 mRNA was not significantly abovethat of background controls (reverse transcriptase). Similarly, viral IEmRNAs (ICP4 and ICP27) were clearly detected in ganglia explanted for 7hrs in the absence but not the presence of TCP.

The data supports the conclusion that MAOIs, such as TCP, inhibit viralIE gene expression and, consequently, reactivation of HSV from latency.

Example 7

This example demonstrates repression of adenovirus E1A expression by theMAO inhibitor TCP.

Methods

Viral infection and treatment with TCP: HeLa cells (2×10⁶) were treatedwith control DMSO (D) or 2 mM Tranylcypromine (T) for 5 hours prior toinfection with various amounts of Adenovirus Type 5 (1.2×10⁸, 2.3×10⁸,and 4.5×10⁸ NAS IU) for 2 or 4 hours. Equal amounts of infected celllysates were resolved by SDS-PAGE and Western blotted with anti-E1A,anti-LSD1, and anti-β tubulin antibodies. HEK293 and uninfected HeLacell lysates represented E1A positive and negative controls.

Results

Similar to the inhibition of the expression of the IE genes of theα-herpesviruses (HSV and VZV), Tranylcypromine also inhibits theexpression of the adenovirus IE protein E1A. In contrast no significantimpact on the levels of the controls LSD1 and β-Tubulin are seen.

Example 8

This Example demonstrates dose-dependent repression of adenovirus E1Aexpression by TCP.

Methods

HeLa cells (2×10⁶) were treated with control DMSO (D) or variousconcentrations of Tranylcypromine (TCP) for 5 hours prior to infectionwith 2.3×10⁹ NAS IU of adenovirus Type 5 for 2 hours. Equal amounts ofinfected cell lysates were resolved by SDS-PAGE and Western blotted withanti-E1A, anti-β Tubulin, and anti-TBP antibodies.

Results

Inhibition of expression of the adenovirus Immediate Early protein E1Aby TCP is seen in the same concentration range as demonstrated for theinhibition of the expression of the α-herpesvirus immediate earlyproteins (FIG. 7). In contrast, no significant impact was seen on thelevels of the control TBP and β-Tubulin proteins.

All references, including publications, patent applications, andpatents, cited herein are hereby incorporated by reference to the sameextent as if each reference were individually and specifically indicatedto be incorporated by reference and were set forth in its entiretyherein.

The use of the terms “a” and “an” and “the” and similar referents in thecontext of describing the invention (especially in the context of thefollowing claims) are to be construed to cover both the singular and theplural, unless otherwise indicated herein or clearly contradicted bycontext. The terms “comprising,” “having,” “including,” and “containing”are to be construed as open-ended terms (i.e., meaning “including, butnot limited to,”) unless otherwise noted. Recitation of ranges of valuesherein are merely intended to serve as a shorthand method of referringindividually to each separate value falling within the range, unlessotherwise indicated herein, and each separate value is incorporated intothe specification as if it were individually recited herein. All methodsdescribed herein can be performed in any suitable order unless otherwiseindicated herein or otherwise clearly contradicted by context. The useof any and all examples, or exemplary language (e.g., “such as”)provided herein, is intended merely to better illuminate the inventionand does not pose a limitation on the scope of the invention unlessotherwise claimed. No language in the specification should be construedas indicating any non-claimed element as essential to the practice ofthe invention.

Preferred embodiments of this invention are described herein, includingthe best mode known to the inventors for carrying out the invention.Variations of those preferred embodiments may become apparent to thoseof ordinary skill in the art upon reading the foregoing description. Theinventors expect skilled artisans to employ such variations asappropriate, and the inventors intend for the invention to be practicedotherwise than as specifically described herein. Accordingly, thisinvention includes all modifications and equivalents of the subjectmatter recited in the claims appended hereto as permitted by applicablelaw. Moreover, any combination of the above-described elements in allpossible variations thereof is encompassed by the invention unlessotherwise indicated herein or otherwise clearly contradicted by context.

1.-11. (canceled)
 12. A method of preventing or treating a viralinfection of a host, comprising administering to the host an effectiveamount of an inhibitor of the protein LSD1.
 13. A method of preventingor treating reactivation of a virus after latency in a host, comprisingadministering to the host an effective amount of an inhibitor of theprotein LSD1.
 14. A method of preventing or treating a viral infectionin a mammal that has undergone, is undergoing, or will undergo an organor tissue transplant, comprising administering to the host an effectiveamount of an inhibitor of the protein LSD1.
 15. The method of claim 12or 13, wherein the inhibitor of the protein LSD1 is administered orallyor topically.
 16. The method of claim 12 or 13, wherein the viralinfection is due to a DNA virus.
 17. The method of claim 16, wherein theDNA virus is a herpesvirus or adenovirus.
 18. The method of claim 17,wherein the herpesvirus is herpes simplex virus type 1, herpes simplexvirus type 2, varicella zoster virus, or cytomegalovirus.
 19. The methodof claim 17, wherein the adenovirus is adenovirus Type 1, 2, 3, 4, or 5.20. The method of claim 12 or 13, wherein the inhibitor of the proteinLSD1 is a monoamine oxidase inhibitor (MAOi).
 21. The method of claim20, wherein the monoamine oxidase inhibitor is tranylcypromine,pargyline, phenelzine, isocarboxazid, or selegiline.
 22. The method ofclaim 12 or 13, wherein the inhibitor of the protein LSD1 is an RNAimolecule.